Fig. 3

ROR2 impairs cell-cycle progression in synchronized cells. A, B Flow cytometry analysis of PI-stained A375 overexpressing ROR2 (A) and M2 cells (B) upon silencing of ROR2. Cells were synchronized and then released by addition of 10% serum as described in “Methods”. The analysis was performed as described in Fig. 1E, F. C, D Effect of ROR2 on the expression of cell-cycle markers. Protein extracts from A375 cells overexpressing ROR2 (C) and M2 cells upon silencing of ROR2 (D) treated as described above were analyzed by western blot with the indicated antibodies. Vinculin was used as a loading control. The upper graphs show the mean ± S.D. of the protein level of each protein at each time-point normalized to the respective loading control. In the lower graphs (line charts), the time-point with the maximal expression was assigned a value of 100 and the relative expression level of the remaining time points were calculated accordingly. The blots displayed are representative of three independent experiments. Statistical significance was tested by one-tailed Student’s T-Test, n = 3. E, F Flow cytometry analysis of Cyclin E expression in A375 overexpressing ROR2 (E) and in M2 (F) upon silencing of ROR2 cells that were treated as described above. Light-grey filled histogram corresponds to control (isotype) antibody and open histograms to Cyclin E antibody. The upper graphs show the mean ± S.D. of Cyclin E MFI at each time-point (from three independent experiments). In the line chart the level of Cyclin E was plotted relative to the time point with the maximal level. Statistical significance was tested by a one-tailed Student’s T-Test. *: p < 0.01, **: p < 0.001, ***: p < 0.0001, n = 3. n.s.: not significant